Plant tissue and cell culture in Vietnam: Forty-five years of development and the future

In this review, the process of formation, development and important achievements as well as the challenges and future prospects of PTCC in Vietnam will be presented. The information is based on works done in Vietnam that have been published in the conference proceedings, scientific journals and prestigious monographs in the country and abroad.

ACADEMIA JOURNAL OF BIOLOGY 2020, 42(3): 53–88 DOI: 10.15625/2615-9023/v42n3.14824 PLANT TISSUE AND CELL CULTURE IN VIETNAM: FORTY-FIVE YEARS OF DEVELOPMENT AND THE FUTURE Nguyen Duc Thanh Institute of Biotechnology, VAST Received 13 February 2020, accepted 28 July 2020 ABSTRACT By 2020, plant tissue and cell culture in Vietnam had undergone 45 years of research and development For nearly half a century, plant tissue and cell culture has been developed to its full potential, especially with the development of genetics, biochemistry and molecular biology It has contributed significantly to basic and practical researches in our country In addition to contributions to domestic science and technology, plant tissue and cell culture in Vietnam has also made impressive imprints in the development of plant tissue and cell culture in the world In this review, I will summarize the process of formation, development and important achievements as well as the challenges and future prospects of this potential field in Vietnam to provide information for researchers, managers, graduate students and other interested readers Keywords: Achievement, contribution, development, plant tissue and cell culture, Vietnam Citation: Nguyen Duc Thanh, 2020 Plant tissue and cell culture in Vietnam: forty-five years of development and the future Academia Journal of Biology, 42(3): 53–88 https://doi.org/10.15625/2615-9023/v42n3.14824 Corresponding author email: nguyenducthanh_pcg@ibt.ac.vn ©2020 Vietnam Academy of Science and Technology (VAST) 53 Nguyen Duc Thanh INTRODUCTION Plant tissue and cell culture (PTCC) includes techniques for culturing plant cells, tissues and organs under aseptic conditions on artificial media with known nutritional components These techniques have been approached by Vietnamese scientists since the 70 s of the twentieth century due to their high potential for research and practical applications Materials used in PTCC include: organs (shoot, root, anthers…), tissues and cells Types of culture include: meristem culture, callus culture, cell culture (single cell, protoplast), embryonic culture, anther culture, pollen culture, thin cell layer culture (TCLC), etc The basic culture methods are: culture on solid medium, culture in liquid medium (static or shaking), culture on semi-liquid medium The main PTCC techniques include: micropropagation (or in vitro propagation), haploid plant production, somatic cell hybridization, embryo rescue, cell line selection and gene transfer PTCC is widely used in micropropagation of agricultural, forestry and flower plants as well as in the conservation of rare or endangered plant species PTCC can be used for screening at the cellular level instead of selecting plants with beneficial traits such as disease resistance and tolerance to adverse environmental conditions Large-scale cultivation of plant cells through liquid culture in bioreactor can generate biomass to obtain secondary substances and recombinant proteins used as biopharmaceuticals Through PTCC, it is possible to create hybrid plants using fusion of protoplasts or rescue embryos of distant hybrid combinations Producing haploid plants from anthersor pollen culture allows the creation of homozygous lines faster in breeding programs Transgenic plants that express genes from animals, bacteria, viruses or other plant genes that lead to production of vaccines, recombinant proteins, plants resistant to insects, viruses and other pathogens, and crops with high nutritional quality In this review, the process of formation, development and important achievements as 54 well as the challenges and future prospects of PTCC in Vietnam will be presented The information is based on works done in Vietnam that have been published in the conference proceedings, scientific journals and prestigious monographs in the country and abroad FORMATION AND DEVELOPMENT Since before 1975, Dr Nguyen Van Uyen, Dr Le Thi Muoi, Le Thi Xuan, Tran Ngoc Cat were the first officials to have an idea to set up a PTCC laboratory in Vietnam After the complete liberation of the South and reunifying of the country (1975), Dr Nguyen Van Uyen and a number of other researchers such as Trinh Manh Dung and Phan Xuan Thanh were assigned to the Vietnam Science Institute in Ho Chi Minh City to set up a PTCC laboratory here, later expanded to Da Lat and other localities Especially, in vitro propagation of potato in “family laboratories” and the production of potato seedlings in pots were implemented by using potato seeding techniques in Da Lat In addition, scientists in the South also studied the propagation of orchids (Huynh Van Hai, Nguyen Van Uyen, 1981; Mai Thi Phuong Hoa et al., 2011; Bui Thi Tuong, Tran Van Minh, 2011), anther culture of rice (Trinh Manh Dung et al., 1986), culture of apical buds of taro plant (Nguyen Thi Quynh & Nguyen Van Uyen, 1985; 1987), protoplast culture (Nguyen Thi Lien Chi, Nguyen Van Uyen, 1989) gene transfer (Nguyen Thi Thanh et al., 1997; Tran Thi Dung & Nguyen Huu Ho, 2003) and research on propagation of some other plants such as coffee (Nguyen Thi Quynh, Nguyen Van Uyen, 1993a; 1993b), Artocarpus heterophyllus Lam (Tran Van Minh, 1997), Polyscias fruticosa (Nguyen Ngoc Dung & Nguyen Van Uyen, 1997), Azadirachta indica (Vu Ngoc Phuong et al., 2000), Paulownia fortune (Seem.) Hermsl (Doan Thi Ai Thuyen et al., 2001) and bamboo species (Vu Ngoc Phuong et al., 2002) In the North, the PTCC research team included four staffs: Dr Le Thi Muoi, Le Thi Xuan, Tran Ngoc Nguyen Hoang Uyen In late 1975 and early 1976, Do Nang Vinh and Plant tissue and cell culture in Vietnam Nguyen Duc Thanh graduated from Azerbaijan State University (in former Soviet Union) have joined the team At that time, working conditions were inadequate and rudimentary; the main building of the Vietnam Scientific Institute was still under construction However, due to news and modernity on PTCC, and the attention of the leaders of the Vietnam Scientific Institute, especially Prof Nguyen Van Hieu, two rooms located on the second floor of the main building (rooms 207 and 208 buildings A2) were urgently completed to serve as plant cell culture laboratory The culture box was originally made of wood with two holes on both sides covered by two cloth tubes, and the inside of the box was fitted with a UV light for sterilization The UV light must turn on at least 30 minutes before the work and the transplanting time should not exceed one hour The culture instruments and test tubes must be brought to the yard of the 1st floor for washing In addition, power and water outages often occur However, even in 1976, the first success of PTCC in Vietnam was marked by the success of culturing rice pollen that was carried out by Do Nang Vinh and Nguyen Duc Thanh In a diary written in Russian dated June 5, 1976 (Figure 1), Nguyen Duc Thanh wrote “Joy has come to us We have received the tiny rice callus that we have been waiting for so long We have been working for a long time with no results And today it has arrived When we grew tiny rice pollen in test tubes, we were certain that we would receive these calli They are small but valuable to us Because of them, we worked hard and waited until from the tiny anthers that inside were pollen grains that could only be seen under a magnification hundreds of times would grow into clumps of cells called “callus” We are very joyful and happy For me, this is the first happiness on the scientific path Oh! How soft, precious and sacred the calli are!” The laboratory led by Dr Le Thi Muoi focused on the research and application of rice and tobacco anther culture techniques (Le Thi Muoi et al., 1978; Le Thi Xuan et al., 1978, 1979; Do Nang Vinh, 1979; Le Tran Binh, 1983; Nghiem Nhu Van, 1989), protoplast culture (Le Thi Muoi & Nguyen Duc Thanh, 1978, Nguyen Duc Thanh, 1983; Le Thi Xuan, 1985), in vitro propagation (potato, carnation, bananas, sugarcane, agave, pineapple, medicinal plants, forestry trees, etc.), cell line selection (Nguyen Hoang Loc et al., 1990), cytoplasmic transfer (Nguyen Duc Thanh et al , 1995, 1997; Le Tran Binh, 1991) and gene transfer (Tran Thi Phuong Lien & Le Xuan Tu, 1993) Beside researching and applying PTCC techniques, the laboratory was also responsible for training researchers and technicians in this field for research institutes, universities and companies such as Agricultural University No (currently Vietnam National University of Agriculture), Institute of Medicinal Materials, Institute of Agricultural Science and Technology (currently Vietnam Academy of Agricultural Sciences), and Cao Bang Tobacco Company, etc Later on, PTCC laboratories were also sat up in the above locations Initially, these PTCC laboratories applied mainly in vitro propagation techniques for multiplication of potato and lily, callus culture and biomass production of medicinal plants, and anther culture to produce double haploid lines in rice In the Central region, after successful defence of PhD thesis at the PTCC laboratory of the Institute of Biology (currently the Institute of Biotechnology), Dr Nguyen Hoang Loc returned to Hue University to setup a PTCC laboratory there His research has focused on propagating a number of plants such as Aquilaria crassma Pierre (Nguyen Hoang Loc et al., 1997), Camellia japonica L (Nguyen Hoang Loc et al., 2001), applying PTCC to produce bioactive substance (Nguyen Hoang Loc et al., 2006) and expression of antigens in plants (Nguyen Hoang Loc et al., 2011) The development of plant tissue and cell culture in Vietnam can be divided into three main stages: 1975−1985: This is the period of formation, approaching and mastering technologies and applying some PTCC techniques such as anther culture to produce 55 Nguyen Duc Thanh double haploid lines and in vitro propagation for rapid propagation of potato, training research staff and technicians on PTCC for universities, research institutes and companies, establishing PTCC laboratories in some universities and research institutes Figure A Nguyen Duc Thanh’s Diary about the first success in Vietnam PTCC dated June 5, 1976; B Callus tissue arising from rice anther culture (Original photograph taken from a 1976 experiment) 1986−1995: This is the period of application and development of PTCC methods and techniques in research and practice, and, at the same time, establishing PTCC laboratories nationwide In addition to in vitro propagation and anther culture techniques, a range of other techniques such as cell line selection, callus and suspension culture for biomass collection, protoplast culture and fusion, embryo rescue, gene transfer have been studied and applied 1996-Present: This is the period to promote the development of biotechnology; in general, genetic engineering and plant cell technology, in particular, for the industrialization and modernization of the country Studies and applications have been focusing on creating resilient crops; fast propagating food crops, vegetables, flowers, 56 fruits, industrial trees and forest trees with good quality, high yield and tolerance to adverse external conditions as well as pests; conservation and development of rare and endangered genetic resources; research on expression of antigen, antibody and recombinant protein, gene transfer, thin cell layer (TCL) culture and photoautotrophic micropropagation ACHIEVEMENTS Basic research The basic research of PTCC in Vietnam mainly focuses on studying the factors affecting callus formation, plant regeneration, growth and development of different species under in vitro conditions, protoplast culture, TCL culture, cytoplasmic genetics, regeneration systems of different plants for Plant tissue and cell culture in Vietnam transgenic transformation and expression of resistance-related genes, genes involved in yield and quality, and the genes encoding antibody and recombinant proteins Le Xuan Tu & Nguyen Thi Xuan Giang (1981, 1982) studied the effects of gamma rays on the development of soybean callus, callus formation and plant regeneration from rice callus The effects of gamma rays on regeneration of rice from mutated calli were also studied by Le Thi Bich Thuy et al (2007) The conditions for culturing callus for biomass production of Maesa balansae Mez were investigated by Quach Thi Lien & Nguyen Duc Thanh (2004) Duong Tan Nhut et al (2009a) studied the effect of coconut water and sucrose on the growth of callus and formation of somatic embryos in Phalaenopsis amabilis (L.) Blume) Influence of culture conditions such as light, CO2 content (Nguyen Tri Minh et al., 2008; Duong Tan Nhut & Nguyen Ba Nam, 2009, Do Thi Gam et al., 2017), sugars (Trinh Thi Lan Anh et al., 2013), growth regulators (Phan Duy Hiep et al., 2014; Vu Thi Lan et al., 2014), amino acids (Tran Trong Tuan et al., 2015), nano silver (Duong Tan Nhut et al., 2017a) in PTCC has been studied for in vitro propagation Le Thi Muoi & Nguyen Duc Thanh (1978) for the first time in Vietnam announced the generation of a complete tobacco plants from protoplast In 1980, Nguyen Duc Thanh & Le Thi Muoi studied the effects of the composition and concentration of plant growth regulators on the process of generating complete tobacco plants from haploid protoplasts Potato protoplasts were also successfully cultured and the complete plants were generated (Nguyen Duc Thanh & Le Thi Muoi, 1981; Nguyen Duc Thanh, 1983, Nguyen Thi Phuong Thao et al., 2012) These results provided an important basis for further research on cytoplasmic transformation, chloroplast genetics and gene transfer In addition, protoplasts of Arabidopsis thaliana (Le Thi Xuan, 1985), Solanum laciniatum (Nguyen Thi Lien Chi & Nguyen Van Uyen, 1989) have also been successfully cultured The use of cell technology in cytoplasmic genetics research, especially the use of protoplasts has achieved impressive results in transferring cytoplasmic male sterility (CMS) into tobacco plants (Nguyen Duc Thanh & Medgyesy, 1988; Le Tran Binh, 1991), transferring whole chloroplasts or chloroplast genes (Nguyen Duc Thanh & Medgyesy, 1989; Nguyen Duc Thanh & Medgyesy, 1993; Nguyen Duc Thanh et al., 1995, 1997; Nghiem Ngoc Minh et al., 1999), creating chloroplast combinations in tobacco by fusion of protoplasts (Nguyen Duc Thanh et al., 1996), and producing cytoplasmic hybrid plants (Nghiem Ngoc Minh et al., 1997) The cytoplasmic genetic research using protoplasts is the unique research area that had only been conducted at the Institute of Biology (currently the Institute of Biotechnology, Vietnam Academy of Science and Technology) from the 80th and 90th of the twentieth century in Vietnam Protoplasts were also used for the gene transfer into Brassica plants (Pham Thi Ly Thu et al., 2001; Pham Thi Ly Thu & Le Huy Ham, 2003) The research team of the Institute of Biology in Da Lat belongs to the Institute of Tropical Biology in Ho Chi Minh City, the National Center for Natural Sciences and Technology (currently the Tay Nguyen Scientific Research Institute, Vietnam Academy of Science and Technology) has studied the method of culturing TCL in lilies (Duong Tan Nhut et al., 2006b) and cauliflower (Duong Tan Nhut & Bui Van The Vinh, 2009) and has been applied to propagate various plants such as lilies, Jatropha curcas, Ngoc Linh ginseng, and orchids (Duong Tan Nhut et al., 2008b; Do Dang Giap et al., 2012a; Vu Thi Hien et al., 2015; Vu Thi Hien et al., 2016; Nguyen Thi Kim Loan et al., 2016) TCL culture was also conducted at the Institute of Agricultural Biology for in vitro propagation of lilies and orchids (Nguyen Quang Thach & Hoang Thi Nga, 2000; 57 Nguyen Duc Thanh Nguyen Phuong Thao & Nguyen Quang Thach 2005; Nguyen Thanh Tung et al., 2010) A very important basic research direction is the research on plant regeneration systems of different plants for gene transfer, as regeneration systems are very important for plant transformation If complete plants cannot be regenerated after the transgenic process, no transgenic crops could be produced Therefore, many plants have been studied to regenerate plants from different tissues for transgenic studies Green bean (Mai Truong et al., 2001), rice (Cao Le Quyen et al., 2011), maize (Nguyen Van Dong et al., 2009; Vu Thi Bich Huyen et al., 2013), peanut (Bui Van Thang et al., 2004; Nguyen Thi Thu Nga & Le Tran Binh, 2012), tomato (Do Xuan Dong et al., 2007), papaya (Le Quynh Lien et al., 2003, Nguyen Minh Hung et al., 2006), Citrus nobilis Loureiro (Do Tien Phat et al., 2007), and Melia azedarach (Do Xuan Dong et al., 2008) regeneration systems were developed for gene transfer purpose Studies on expression of genes related to plant tolerance, yield and quality, and genes for antigen and recombinant proteins have been studied through PTCC in a number of laboratories in the Institute of Biotechnology, Institute of Tropical Biology, Institute of Agricultural Genetics, etc NAC2 gene from L12 peanut cultivar coding for protein related to drought tolerance was expressed in tobacco plants (Nguyen Thi Thu Nga et al., 2015) Other genes related to drought tolerance in plants such as GmHK06, GmRR34, and transcription factors like GmNAC092, GmNAC083, GmNAC057 were successfully expressed in soybeans (Hoang Thi Lan Xuan et al., 2015; Nguyen Binh An Thu et al., 2015) Insect resistance gene cryIA(c) was expressed in tobacco (Huynh Thi Thu Hue et al., 2008) Many studies have been conducted to express antigens in plants such as: VP2 antigen gene that induces an immune response in chickens in duckweed (Le Huy Ham et al., 2009), HA antigen of H5N1 virus in soybean seed (Nguyen Thu Hien et al., 2013), antigen GP5 of PRRS virus in tobacco (Dao Thi Sen 58 et al., 2016), H7 antigen of influenza A/H7N9 virus in Nicotiana benthamiana (Le Thi Thuy et al., 2017) Recombinant protein interleukin7, a major regulator of the human immune system (Nguyen Huy Hoang et al., 2017), rabies glycoprotein (Le Quynh Lien et al., 2008) and protein M of PRRS virus causing blue ear disease (Nguyen Thi Minh Hang et al., 2018) were, successfully, expressed in Nicotiana plants HIV-1-p24 gene, also, expressed in tomato-Lycopersicon esculentum Mill (Phan Duc Chi et al., 2013) Practical application Since the beginning of the approach to PTCC techniques, Vietnamese researchers have focused on the practice of producing pure lines, insect-resistant and disease-resistant, and tolerant to adverse environmental factors (salinity, drought) as well as high yield and high quality plant varieties In particular, PTCC has been widely applied to the rapid propagation of agricultural, forestry, flower and medicinal plant species through stem cutting technique, apical shoot-tip culture, culture of somatic embryos, artificial seeds, etc Researchers were also interested in the use of PTCC in biomass production to obtain bioactive substances Production of double haploid lines through anther culture The haploid plants from pollen have great practical significance, because haploid plants are ideal materials to create a pure line Double haploid lines can be produced by colchicine treatment of haploid plants or through haploid callus tissue culture Purebred lines are particularly significant in production of hybrids between incompatible parents and shortening the breeding time The production of haploid plants through anther culture has been widely used in producing double haploid lines in Vietnam Double haploid rice (Le Thi Muoi et al., 1978) and tobacco plants (Le Thi Xuan et al., 1978) from in vitro anther culture were the first success of PTCC in Vietnam These works have laid the foundation for the production of double haploid rice varieties, contributing to Plant tissue and cell culture in Vietnam shortening the time for rice breeding (Bui Ba Bong et al., 1997; Nghiem Nhu Van et al., 2002, 2004; Phan Thi Bay et al., 2004c) The two VH1 and VH2 rice lines created by the anther culture method have been varietal tested and have gone for trial production (Nghiem Nhu Van et al., 2006) Blast resistant HPMD4, HPMD6, HPMD9, HPMD13, HPMD20 and good quality HPMD4, HPMD9 rice lines have been created by culturing F1 anthers of a hybrid between quality and blast resistant rice varieties (Phan Thi Bay et al., 2004c) Anther culture was also applied to create pure lines for restoring the quality of specialty Tu Le sticky rice (Dang Thi Minh Lua et al., 2009) (Figure 2) Figure Growing Tu Le sticky rice that was improved by anthers culture in Tu Le commune, Yen Bai Province Producing disease-resistant potatoes and quality oranges through protoplast fusion Due to the absence of cellulose wall, protoplasts can be fused to produce somatic hybrids, including nuclear hybrid (Do Thi Thu Ha et al., 2012) and cytoplasmic hybrid (Nguyen Duc Thanh et al., 1996) The use of protoplasts in plant breeding practice has been carried out by the research team of the Institute of Agricultural Biology, University of Agriculture #1 By fusing protoplasts of the cultivated potato (Solanum tuberosum L (2n = 4x = 48) and the wild potato species (Solanum bulbocastanum, Solanum tarnii, Solanum pinnatisectum (2n = 2x = 24)) with the ability resistant to late blight Related to this, Hoang Thi Giang et al (2013, 2014) have created a number of late blight resistant potato lines At the Agricultural Genetics Institute, in order to improve the quality of orange varieties, Ha Thi Thuy et al (2010) fused protoplasts between the local orange variety (Citrus nobilis) and sweet orange varieties (C sinensis) Producing plants resistant to salinity, drought and pests through selection of cell lines Selection of plant cell lines is based on heterogeneity of tissues and cells in in vitro culture resulting in somatic variation In Vietnam, the selection of plant cell lines was applied to select the salinity, drought and disease resistant lines Nguyen Hoang Loc et al (1990) selected NaCl-tolerant tobacco lines and drought tolerant sugarcane lines (Nguyen Hoang Loc et al., 2003) through callus culture Truong Thi Bich Phuong et al (2002) reported the selection of drought tolerant rice lines by callus culture Vu Thi Thu Thuy et al (2013) selected drought tolerant groundnut lines from dehydrated calli By selecting cell lines from radiation treated calli, Nguyen Thi Huong et al (2017) have selected a saline tolerant Eucalyptus urophyla callus line that can be regenerated on medium with 125 mM NaCl Le Thi Bich Thuy et al (1997) reported the selection of rice lines resistant to fungal pathogen (Piricularia oryzae) and subsequently created rice lines resistant to blast disease (Phan Thi Bay et al., 1997) Rice varieties DR1, DR2 were created by selecting dehydrated calli (Dinh Thi Phong et al., 1999), and DR2 has been recognized as a national variety Producing transgenic plants resistant to drought, pests, diseases, and increasing productivity and quality In addition to the basic research orientation as described above, some laboratories have made efforts to create transgenic plant with drought tolerance (Cao Le Quyen et al., 2009), disease-resistance (Vu Thi Lan et al., 2017), increased productivity 59 Nguyen Duc Thanh (Nguyen Duc Thanh et al., 2015) and quality transgenic crops (Tran Thi Luong et al., 2014; Tran Thi Luong, Nguyen Duc Thanh, 2015) However, the results are still modest MtOsDREB2A (Cao Le Quyen et al., 2009), OsNLI-IF (Nguyen Duy Phuong et al., 2015) and OsNAC1 (Pham Thu Hang et al., 2016) control drought tolerance were transferred to rice and NF-YB2 (Nguyen Van Dong et al., 2015) gene was transferred to maize to creating drought tolerant lines The gene coding glycine-betaine was transferred to Melia azedarach L to create salt tolerant plants (Chu Hoang Ha, Bui Van Thang, 2017) Herbicide resistant (Pham Thi Ly Thu et al., 2015) and insect resistant maize (Pham Thi Ly Thu et al., 2013), insect resistant soybean (Nguyen Van Dong, 2012) were created by transferring GA21, cryA1, cry1b genes into maize and soybean Tran Thi Cuc Hoa et al (2017) used the new vector pHOA60, pH0A100, pH0A130 to transfer VIP3A gene into soybean to create plants resistant to fruit flies Transgenic tobacco plants resistant to two mosaic viruses were created by RNAi transformation (Pham Thi Van et al., 2009) Also by RNAi transfer method, Nguyen Thi Hai Yen et al (2011) created a line of transgenic tomato PT18 resistant to viral leaf curl disease Vu Thi Lan et al (2018) transferred the cry3CA1 gene to sweet potato to create sweet potato lines resistant to the Cylas formicarius Vi Thi Xuan Thuy et al (2016, 2017) reported the transformation of the DEFENCIN (ZmDEF1) gene from the local maize resistant to the weevil to the elite maize variety to create elite maize lines resistant to weevil Shrunken (Sh2) (Tran Thi Luong et al., 2014) and Brittle (Bt2) genes (Nguyen Thi Thu et al., 2014; Nguyen et al., 2016) encoding ADP-glucose pyrophosphorylase-an enzyme that regulates starch synthesis, were successfully transferred into maize and transgenic maize that increase starch content from 10.12 to 16.04% and yield over tons/ha were obtained As maize is a low-quality food crop (lack of lysine, tryptophan, low provitamin A including α-carotene, β-carotene 60 and β-cryptoxanthin), with the aim of improving the quality of maize, Tran et al (2017) had transferred the IbOr gene from the Hoang Long sweet potato variety into several maize lines and created transgenic maize plants with increased -carotene content more than 10 fold, contributing to improving the nutritional quality of maize (Tran et al., 2017; Tran Thi Luong & Nguyen Duc Thanh, 2018) Tran Thi Xuan Mai & Tran Thi Cuc Hoa (2017) reported the production of transgenic rice plants with lysine content increased up to 38% by transferring DHDPS-r1 gene encoding the dihydrodipicolinate synthase (DHDPS) enzyme into the Taipei 309 rice variety Rapid propagation, conservation and development of genetic resources In vitro propagation, in vitro micropropagation, or simply micropropagation has the advantage of having a high multiplication coefficient, the ability to multiply large numbers of plants in a small area Disease-free plants and no contact with disease sources should ensure seedlings are free of diseases In addition, micropropagation makes it easy to exchange and transport the seedlings Therefore, micropropagation has been widely applied in plant breeding, conservation and development of rare genetic resources During the 45 years of PTCC development in Vietnam, perhaps the most important contribution is the in vitro propagation of agricultural, forestry, floral and medicinal plants, including some endangered plants Agricultural plants Among the micropropagated agricultural plants, potato was the first and obtained impressive results Nguyen Van Uyen had successfully implemented in vitro propagation of potato in Da Lat at the family laboratories and produced potato seedlings in pots by using potato seedbed method In addition to propagating potato by cutting in vitro stems, propagation through producing minitubers from in vitro seedlings has also been conducted successfully (Nguyen Quang Thach et al., 2005) Plant tissue and cell culture in Vietnam After potato, banana (Vu Ngoc Phuong et al., 2009; Do Dang Giap et al., 2012b), pineapple (Phan Thi Bay et al., 1994a; Nguyen Duc Thanh et al., 2003), sugarcane (Ha Thi Thuy et al., 1998; Nguyen Kim Lan et al., 1998) have been propagated in vitro and provided millions of qualified and disease-free seedlings for breeding The Institute of Tropical Biology, the Institute of Biotechnology, the Agricultural Genetics Institute, the Vegetable Research Institute, the Agricultural University #1 are the places that have propagated bananas, especially Cavendish species in the large scale Vu Ngoc Phuong et al., (2009) reported about in vitro propagation of banana (Cavendish sp.) on an industrial scale The Cayenne pineapple propagation protocol has been developed by the Institute of Biotechnology in cooperation with the Institute of Fruit and Vegetable Research and was transferred to seedling production to supply pineapple seedlings for farms (Nguyen Duc Thanh et al., 2003) (Figure 3) Figure In vitro propagated Cayene pineapple grown at Suoi Hai Farm (in 2000) In vitro propagation of sugarcane has been carried out methodically at the Institute of Agricultural Genetics, especially the development and propagation of new and high yielding sugarcane at the industrial scale (Ha Thi Thuy et al., 1998, 1999., 2000a, 2000b) Flower Micropropagation has made a significant contribution to the propagation of flowering plants, especially orchids PTCC laboratories throughout the North, Central and South have studied and propagated these precious flowers Tay Nguyen Institute of Biology (current Tay Nguyen Scientific Research Institute) propagates Cymbidium sp (Phan Xuan Huyen et al., 2004), Paphiopedilum callosum (Vu Quoc Luan et al., 2012, 2013b), Paphiopedilum graxtrixianum (Vu Quoc Luan et al., 2013a), and Dendrobium heterocarpum Lindl (Dang Thi Tham et al., 2018) The Institute of Tropical Biology propagated Dendrobium sp., Phalaenopsis sp., Cymbidium sp and Rhynchostylis sp (Mai Thi Phuong Hoa et al., 2011; Bui Thi Tuong Thu, Tran Van Minh, 2011) The number of orchids like Phalaenopsis Sogo Yukidian (Nguyen Thi Son et al., 2014), Dendrobium fimbriatum, Dendrobium nobile L (Nguyen Thi Son et al., 2012a, 2012b; Vu Ngoc Lan & Nguyen Thi Ly Anh, 2013), Cymbidium sp (Nguyen Quang Thach et al., 2004), Cymbidium iridioides (Hoang Thi Nga et al., 2008, and Paphiopedilum hangianum Perner & Gruss (Hoang Thi Giang et al., 2010) were successfully propagated in the Institute of Agricultural Biology Pham Thi Kim Hanh et al (2009) in the Agriculture Genetics Institute reported the propagation of Rhynchostylis gigantean orchid Other orchids such as Dendrobium anosmum (Nguyen Quynh Trang et al., 2013), Dendrobium crepidatum Lindl & Paxt (Nguyen Van Ket & Nguyen Van Minh, 2010), and Dalybium gratiosisstimum Reichenb F (Vu Kim Dung et al., 2016), have also been propagated through micropropagation In addition to orchids, in vitro propagation of many other flowering plants have been studied e.g Lilium longiflorum (Nguyen Thi Phuong Thao & Nguyen Quang Thach, 2005; Nguyen Thi Phuong Thao et al., 2006; Doan Thi Quynh Huong et al., 2013), Lilium spp (Nguyen Thi Ly Anh et al., 2005; Do Minh Phu et al., 2009), Polianthes tuberosa (Duong Tan Nhut, 1994), carnation (Nghiem Ngoc Minh, 1992), roses (Phan Thi Bay et al., 1996; Nguyen Thi Kim Thanh, 2005; Nguyen Thi 61 Nguyen Duc Thanh Phuong Thao et al., 2015, Nguyen Van Viet, 2017), Chrysanthemums (Nguyen Thi Dieu Huong & Duong Tan Nhut, 2004), Anthurium andraeanum (Hoang Thi Nhu Phuong et al., 2014; Nguyen Thi Thuy Diem, 2015), and Hydrangea macrophylla (Thi The Luc et al., 2017) have been successfully propagated Forest trees Among forestry trees, acacia has been the most widely and successfully used for in vitro propagation Particularly, hybrid Acacia, Acacia crassicarpa A Cunn Ex Benth, and Acacia auriculiformis A Cunn Ex Banth (Doan Thi Mai et al., 1998, 2009b; Phi Hong Hai & Van Thu Huyen, 2016; Trieu Thi Thu Ha et al., 2014) Eucalyptus varieties (Le Kim Dao, 2001) have been widely propagated to provide seedling sources for reforestation Besides, other species such as Melaleuca (Phung Thi Hang & Nguyen Bao Toan, 2011), Caribaea pine (Kieu Phuong Nam et al., 2009), resin pinePinus merkusii (Do Tien Phat et al., 2009; Pham Thi My Lan, Nguyen Xuan Cuong, 2014), Paulownia fortunei (Nguyen Thi Quynh et al., 2002; Doan Thi Ai Thuyen et al., 2001), Aquilaria crassma Pierre (Nguyen Hoang Loc et al., 1997), Chukrasia tabularis (Doan Thi Mai et al., 2009a), Aquilaria crassna Pierre (Le Van Thanh & Nguyen Thi Hien, 2010), Azadirachta indica A Juss (Vu Ngoc Phuong et al., 2000) Sinocalamus latiflorus and Dendrocalamus asper (Vu Ngoc Phuong et al., 2002) were also propagated by the micropropagation method Medicinal plants For medicinal plants, many species have been cultivated and propagated in vitro However, the results are still very modest The plant species that have been studied and propagated include Crinum latifolium, Polyscias fruticosa, Morinda officinalis, Anoectochilus setaceus Blume, Dendrobium officinale Kimura et Migo, and ginseng species Quach Thi Lien et al (2003) studied regeneration of Crinum latifolium plants Polyscias fruticosa was most studied for in 62 vitro propagation (Nguyen Ngoc Dung, Nguyen Van Uyen, 1997; Vu Hoai Sam & Pham Van Hien, 2005; Le Nhu Thao et al., 2014; Dao Duy Hung et al., 2017; Trinh Viet Nga et al., 2019) Orchid species with high pharmaceutical value such as Anoectochilus setaceus Blume or Anoectochilus roxburghii (Wall.) Lindl (Nguyen Quang Thach & Phi Thi Cam Mien, 2012; Truong Thi Bich Phuong & Phan Ngoc Khoa, 2013; Vu Quoc Luan et al., 2015; Tran Thi Hong Thuy et al., 2015; Phan Xuan Huyen et al., 2018), Dendrobium officinale Kimura et Migo (Trinh Thi Thuy An & Nguyen Thi Tam, 2017; Le Thi Diem & Vo Thi Bach Mai, 2017) have been, successfully, in vitro propagated Other medicinal plants like Codonopsis javanica Blume (Phan Xuan Huyen et al., 2014; Doan Trong Duc et al., 2015), Milletia speciora Champ (Ta Nhu Thuc Anh et al., 2014), Celastrus hindsii (Ta Nhu Thuc Anh & Nguyen Thi Bich Thu, 2012), Artemisia annua (Mai Thi Phuong Hoa et al., 2012; Bui Thi Tuong Thu et al., 2012), Japanese Angelica acutiloba Kitagawa (Hoang Ngoc Nhung & Nguyen Thi Quynh, 2015), Lavandula angustifolia (Do Tien Vinh et al., 2016), Rehmannia glutinosa (Vu Hoai Sam et al., 2018), and Polygonum multiflorum (Truong Thi Bich Phuong et al., 2008; Bui Van Thang, 2017) have also been propagated in vitro Ginseng, in particular, Ngoc Linh ginseng (Figure 4) have received much attention for in vitro propagation Ngoc Linh ginseng (Panax vietmamensis Ha et Grushv) has been propagated through somatic embryos from leaves, petioles or stems (Mai Truong et al., 2013; Vu Thi Hien et al., 2014), by artificial seeds (Tran Thi Huong & Duong Tan Nhut, 2011 ) or in vitro root generation (Hoang Xuan Chien et al., 2011) In addition, other ginseng species such as Lai Chau ginseng - Panax vietnamensis var fuscidiscus (Le Hung Linh & Dinh Xuan Tu, 2017), Hibiscus sagittifolius Kurz (Phan Duy Hiep et al., 2014) and Milletia speciora Champ (Ta Nhu Thuc Anh et al., 2014) have also been studied for in vitro propagation Nguyen Duc Thanh An efficient protocol for plant regeneration in papaya (Carica papaya L.) via multi-shoot induction Journal of Biotechnology, 1(3): 355–362 (in Vietnamese with English summary) Le Quynh Lien, Phan Trong Hoang, Do Xuan Dong, Chu Hoang Ha, Le Tran Binh, 2008 Expression of rabies virus glycoprotein in 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